Methods and compositions for bone and cartilage repair

ABSTRACT

Biologically active extract of  Hibiscus tiliaceus  L. and compositions containing the extract are described. Therapeutic uses of the extract and compositions include promoting healing of bone and cartilage injuries and also the promotion of bone and cartilage formation.

PRIORITY CLAIM

The present application is a continuation of U.S. patent applicationSer. No. 13/514,249 filed Nov. 19, 2012, which is a National Stage ofInternational Application No. PCT/AU2010/001679 filed Dec. 10, 2010,which claims priority to Australian Patent Application No. 20009906034filed Dec. 10, 2009, the entire contents of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the use of plant extracts andcompositions comprising the same for the treatment of bone and cartilageinjuries, diseases or defects, the promotion of healing of bone andcartilage injuries, and the promotion of bone and cartilage formation.Particular embodiments as disclosed herein find application in, interalia, the treatment of bone fractures and in bone formation andregeneration.

BACKGROUND

Bone is a living tissue comprising a number of constituents includingcalcium carbonate, calcium phosphate, collagen and water and iscontinuously being replenished by resorption and deposition of bonematrix.

When a bone fractures the usual treatment is to reposition the fracturedbone back into place, to stabilise the position of the bone, and then towait for the bone healing process to occur. Bone healing is a complexprocess which is generally regarded as involving three phases: reactivephase, reparative phase and remodelling phase. During the reactive phasea haematoma forms at the fracture site and inflammatory cells andfibroblasts infiltrate the bone under prostaglandin mediation. Thisresults in the formation of granulation tissue, ingrowth of vasculartissue, and migration of mesenchymal cells. During the reparative phase,fibroblasts begin to lay down stroma that helps support vascularingrowth. As vascular ingrowth progresses, a collagen matrix is laiddown while osteoid is secreted and subsequently mineralised, which leadsto the formation of a soft callus (cartilage) around the repair site.After a period of time from weeks to months the cartilage ossifies,forming a bridge of woven bone between the fracture fragments. Duringthe remodelling phase the healing bone is restored to its originalshape, structure, and mechanical strength. The remodelling phase occursslowly over months to years, however, adequate bone strength istypically achieved in three to six months.

The healing of bone fractures is clearly a lengthy process. Notably,cartilage, unlike other connective tissues, does not contain bloodvessels and therefore compared to other connective tissues, grows andrepairs more slowly. Further, bone healing can be delayed or impairedwhen any of the healing processes do not function properly or in atimely manner. This can be both a major setback to the individual and acostly clinical problem. Accordingly, approaches that speed up thebody's natural process of regenerating bone and cartilage are clearlyadvantageous.

The present inventors have surprisingly found that plant extractsdisclosed herein and compositions comprising same, promote bone andcartilage repair by inducing new bone formation and new cartilagegrowth.

SUMMARY

According to a first aspect of the invention there is provided abiologically active extract of Hibiscus tiliaceus L. The extract mayfurther comprise an extract of one or more of Vigna marina (Burm.)Merr., Cocos nucifera L., or Terminalia catappa L.

The extract(s) may be derived from one or more of the following: bark,leaf, vine, bean, husk or nut. In one embodiment the Hibiscus tiliaceusL. extract is derived from bark, typically fresh crushed bark.

The extract may be prepared using and/or comprises, a plant based oil, ahydrocarbon and/or an alcohol. The plant based oil may be derived fromplant seeds or fruit. In a particular embodiment the plant based oil isderived from Cocos nucifera. The Cocos nucifera oil may be, for example,virgin Cocos nucifera oil, refined Cocos nucifera oil, hydrogenatedCocos nucifera oil or fractionated Cocos nucifera oil.

According to a second aspect of the invention there is provided acomposition comprising an extract according to the first aspect, and oneor more pharmaceutically acceptable carriers, diluents and/orexcipients.

In particular embodiments, the composition is for the treatment of boneor cartilage injury and/or the promotion of bone or cartilage formation,repair or regeneration.

The composition may be formulated so as to be suitable foradministration by any route, for example topical or parenteral.Parenteral administration may comprise, for example, intraosseousinfusion or intrathecal injection.

According to a third aspect of the invention there is provided a methodfor promoting the formation, repair or regeneration of bone or cartilagein a subject, the method comprising administering to the subject in needthereof an effective amount of an extract according to the first aspect,a composition according to the second aspect, or osteoblasts,osteoblastic progenitor cells, or bone segments or fragments cultured inthe presence of said extract or composition.

The subject may be suffering from a bone or cartilage injury, defect ordisease or may be susceptible or predisposed to such an injury, defector disease. The injury may be, for example, a bone fracture. The diseasemay be a degenerative bone disease, for example, osteoporosis. Thesubject may be undergoing or have undergone a bone or cartilage graftprocedure, such as a skeletal fusion procedure, for example a spinalfusion procedure.

The osteoblasts or osteoblastic progenitor cells may be autogeneic,allogeneic or xenogeneic.

According to a fourth aspect of the invention there is provided a methodfor treating a bone or cartilage injury, defect or disease in a subject,the method comprising administering to the subject an effective amountof an extract according to the first aspect, a composition according tothe second aspect, or osteoblasts, osteoblastic progenitor cells, orbone segments or fragments cultured in the presence of said extract orcomposition.

The injury may be, for example, a bone fracture. The disease may be adegenerative bone disease, for example, osteoporosis. The extract orcomposition may enhance the rate or extent of healing of the injury ordisease. The extract or composition may promote bone or cartilageformation, repair or regeneration.

According to a fifth aspect of the invention there is provided a methodfor promoting bone or cartilage growth surrounding a bone or cartilagegraft or implantable device, the method comprising administering to asubject prior to, during or after a graft or implantation procedure, aneffective amount of extract according to the first aspect, a compositionaccording to the second aspect, or osteoblasts, osteoblastic progenitorcells or bone segments or fragments cultured in the presence of saidextract or composition.

The graft may be an autograft or allograft. The graft procedure may be,for example, for the repair of a bone fracture or for the purposes ofskeletal fusion such as spinal fusion. The implantable device may be abone fixation device or a prosthesis. The extract according to the firstaspect, the composition according to the second aspect, or osteoblasts,osteoblastic progenitor cells or bone segments or fragments cultured inthe presence of said extract or composition may be coated on to theprosthesis, used as an adjunct to a bone fixation device, or used as anadjunct to a bone cement used to anchor the prosthesis or bone fixationdevice.

In accordance with the above aspects and embodiments the administrationto the subject may be during an arthroscopic or open surgical procedure.

According to a sixth aspect of the invention there is provided a methodfor the formation or growth of bone, the method comprising incubatingosteoblasts, osteoblastic progenitor cells or bone segments or fragmentsin the presence of an effective amount of an extract according to thefirst aspect or a composition according to the second aspect, underconditions suitable to induce the formation or growth of bone.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described, by way of non-limitingexample only, with reference to the accompanying drawings in which:

FIGS. 1A-1D show histologic profiles of skin of 10 to 14 weeks oldfemale rats. FIG. 1A, no treatment; FIG. 1B, daily application for 7days of coconut oil; FIG. 1C, daily application for 7 days of Hibiscustiliaceus L. in ethanol; and FIG. 1D, daily application for 7 days ofHibiscus tiliaceus L. in coconut oil. The skin samples were stained withhematoxylin and eosin and are shown at ×400 magnification.

FIGS. 2A-2F show histologic profiles of skin of 12 week old female NewZealand rabbits. FIGS. 2A and 2B, no. treatment; FIGS. 2C and 2D, dailyapplication for 7 days of a combination extract of Vigna marina (Burm.)Merr., Cocos nucifera L., Terminalia catappa L. and Hibiscus tiliaceusL. in coconut oil; FIGS. 2E and 2F, daily application for 7 days ofHibiscus tiliaceus L. in coconut oil. The skin samples were stained withhematoxylin and eosin and are shown at ×40 (FIGS. 2A, 2C, and 2E) and×400 (FIGS. 2B, 2D, and 2F) magnification.

FIGS. 3A-3L show histologic differences in untreated (FIGS. 3A and 3B)and treated (FIGS. 3C to 3F) 18 month old oestrogen deficient (ovariesremoved at age 6 weeks) rats; and untreated (FIGS. 3G to 3H) 18 monthold non-oestrogen deficient (control surgical procedure carried out atage 6 weeks and rats allowed to recover) and treated rats (FIGS. 3I to3L), following a surgically created fracture of the femur by osteotomy,and operative repair with internal fixation. The treated rats weresubjected to a daily application of an extract of Hibiscus tiliaceus L.in coconut oil to the fracture site for 21 days. The fracture histologysamples were stained with hematoxylin and eosin (FIGS. 3A, 3C, 3E, 3G,3I, and 3K) and Saffron-0 Stain (FIGS. 3B, 3D, 3F, 3H, 3J, and 3L) whichstains proteoglycan in cartilage purple; and are shown at ×12magnification.

FIG. 4 shows a graph of histological scoring at the fracture sites inuntreated 18 month old oestrogen deficient (ovaries removed at age 6weeks) rats (Ovx Control) (control surgical procedure was carried out atage 6 weeks and rats allowed to recover); and treated 18 month oldoestrogen deficient (ovaries removed at age 6 weeks) rats (Ovx rx) rats,following a surgically created fracture of the femur by osteotomy, andoperative repair with internal fixation. The treated rats were subjectedto a daily application of an extract of Hibiscus tiliaceus L. in coconutoil to the fracture site for 3 weeks.

FIGS. 5A-5D show computer models (FIGS. 5A and 5C) and radiographs(FIGS. 5B and 5D) of surgically created 20 mm defects of the ulna(including periosteum) by osteotomy, and with no graft or substitutefiller in 12 week old NZ white rabbits, following a daily applicationfor 7 days to the epithelial surface of the back, not on the limb of thefracture site of: FIGS. 5A and 5C; coconut oil with intermediatehydrocarbon Extract; and FIGS. 5C to 5D; hydrocarbon extract of Hibiscustiliaceus L. in coconut oil.

FIGS. 6A-6F show histologic profiles in 12 week old female New Zealandrabbits following a daily application for 7 days to the epithelialsurface at the fracture site (surgically created 20 mm defect of theulna (including periosteum) by osteotomy, and with no graft orsubstitute filler) of: a combination extract of Vigna marina (Burm.)Merr., Cocos nucifera L., Terminalia catappa L. and Hibiscus tiliaceusL. in coconut oil (FIGS. 6C and 6D); an extract of Hibiscus tiliaceus L.in coconut oil (FIGS. 6E and 6F). FIGS. 6A and 6B, untreated controls.The samples were stained with pentachrome and are shown at ×12.5 (FIGS.6A, 6C, and 6E) and ×25 (FIGS. 6B, 6D, and 6F) magnification.

FIGS. 7A-7H show histologic profiles in 12 week old female New Zealandrabbits following a daily application to the epithelial surface at thefracture site (surgically created surgically created 20 mm defect of theulna (including periosteum) by osteotomy, and with no graft orsubstitute filler) for 7 days of: FIGS. 7A and 7B, a combination extractof Vigna marina (Burm.) Merr., Cocos nucifera L., and Terminalia catappaL. (no Hibiscus tiliaceus L.) in coconut oil; FIGS. 7C and 7D, anextract of Hibiscus tiliaceus L. in ethanol; FIGS. 7E and 7F, an extractof Hibiscus tiliaceus L. in coconut oil following extraction in ethanol;and FIGS. 7G and 7H, an extract of Hibiscus tiliaceus L. in coconut oilfollowing extraction in ethanol and hydrocarbon. The samples werestained with pentachrome and are shown at ×12.5 magnification.

FIG. 8 shows a graph of the bone growth score in surgically created 20mm defects of the ulna (including periosteum) by osteotomy, in 12 weekold NZ white rabbits, following a daily application for 7 days to theepithelial surface at the fracture site of: Hibiscus tiliaceus L.precipitate of intermediate density in ethanol (HeA Intermediate);Hibiscus tiliaceus L. in coconut oil following extraction with ethanol(eAo); Hibiscus tiliaceus L in ethanol (eA Ethanol); and a combinationextract of Vigna marina (Burm.) Merr., Cocos nucifera L., and Terminaliacatappa L. in coconut oil following extraction with ethanol (eNPKo).

FIGS. 9A-9E show x-rays (FIGS. 9A to 9D) and a CT scan (FIG. 9E) of thespine of female New Zealand rabbits which underwent bilateral singlelevel postero-lateral fusion using autograft harvested from the iliaccrest at 12 weeks old. Surgery was followed by routine care (FIGS. 9Aand 9B) or daily application for 5 days and then twice weeklyapplication to 6 weeks to the epithelial surface rostral to, but notoverlying the fracture site of an extract of Hibiscus tiliaceus L. incoconut oil following extraction in ethanol and hydrocarbon (FIGS. 9C to9E). X-Rays show the bilateral spinal repair, with FIG. 9A the untreatedand FIG. 9C the treated 6 week post operative healing fusion mass, andthe donor harvest site on the iliac crest in untreated (FIG. 9B) andtreated (FIG. 9D) animals at 6 weeks. The CT scan shows the iliac crestof a treated animal at 6 weeks (FIG. 9E).

FIGS. 10A and 10B show histologic profiles of the spine of female NewZealand rabbits which underwent bilateral single level postero-lateralfusion using autograft harvested from the iliac crest at 12 weeks old.Surgery was followed by routine care (FIG. 10A) or daily application for5 days and then twice weekly application to 6 weeks to the epithelialsurface rostral to, but not overlying the fracture site of 5 ml of anextract of Hibiscus tiliaceus L. in coconut oil following extraction inethanol and hydrocarbon (FIG. 10B). The samples were stained withhematoxylin and eosin and are shown as a composite of images at ×12.5magnification showing the transverse process and the spinal fusion mass2-3 mm lateral to the vertebral body.

FIG. 11 shows the results of mechanical testing of treated autografts at8 weeks and untreated controls at 12 weeks.

FIGS. 12A and 12B show mass chromatograms of: FIG. 12A, Hibiscustiliaceus L. in coconut oil following extraction in ethanol andhydrocarbon and FIG. 12B, the hydrocarbon intermediate layer.

DETAILED DESCRIPTION

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

As used herein the terms “effective amount” and “effective dose” includewithin their meaning a non-toxic but sufficient amount or dose of anextract or composition' to provide the desired effect. The exact amountor dose required will vary from application to application and subjectto subject depending oh factors such as whether the extract orcomposition is administered in vitro, ex vivo or in vivo, the speciesbeing treated, the age and general condition of the subject, theseverity of the condition being treated, the particular extract orcomposition being administered and the mode of administration and soforth. Thus, it is not possible to specify an exact effective amount ordose. However, for any given case, an appropriate effective amount ordose may be determined by one of ordinary skill in the art using onlyroutine experimentation.

As used herein the term “extract” refers to an active preparationderived from one or more plants. In the context of the specification by“active” it is meant that the extract is capable of producing a desiredtherapeutic benefit as disclosed herein. An extract is obtained by aprocess of “extraction” which will be understood by those skilled in theart as, in general terms, comprising treating plant material with asolvent, a liquid, or a supercritical fluid to dissolve the activepreparation and separate the same from residual unwanted plant material.An extract may be in liquid form (for example as a decoction, solution,infusion or tincture) or solid form (for example as a powder orgranules). The term “combination extract” as used herein refers to anextract prepared from more than one plant species. In a combinationextract the plant material from each of the plant species may besubjected to the extraction process together or separately. That is,material from some or all of the species may be combined prior toaddition of the solvent, liquid or supercritical fluid, and/or materialfrom some or all of the species may be independently treated with asolvent, liquid or supercritical fluid and the preparations so obtainedare subsequently combined. As such, the same or different solvents (orliquids or supercritical fluids) may be used to extract the activepreparation from the different species. The terms “extract” and“combination extract” may be used interchangeably throughout thespecification.

The terms “promoting”, “promotion” and variations thereof as used in thecontext of bone or cartilage formation, repair and regeneration refer tothe ability of an extract or composition as disclosed herein to induce,stimulate, enhance, or otherwise effect or cause, either directly orindirectly, the formation of new bone or cartilage, or the repair orregeneration of bone or cartilage. The promotion may occur in vivo, exvivo or in vitro. The terms “enhancing”, “enhancement” and variationsthereof as used herein in the context of bone or cartilage formation,repair and regeneration refer to the ability of an extract orcomposition as disclosed herein to enhance or increase, either directlyor indirectly, the rate or degree of action of natural physiologicalprocess(es) involved in bone or cartilage formation, repair and/orregeneration.

As used herein the term “subject” includes humans, primates, livestockanimals (eg. sheep, pigs, cattle, horses, donkeys), laboratory testanimals (eg. mice, rabbits, rats, guinea pigs), companion animals (eg.dogs, cats), show animals (eg. dogs, cats, pigs, cattle, sheep, horses)and captive wild animals (eg. foxes, kangaroos, deer). Typically, thesubject is human or a laboratory test animal. Even more typically, thesubject is a human.

As used herein the terms “treating”, “treatment”, “preventing” and“prevention” refer to any and all uses which remedy a condition orsymptoms, prevent the establishment of a condition or disease, orotherwise prevent, hinder, retard, or reverse the progression of acondition or disease or other undesirable symptoms in any waywhatsoever. Thus the terms “treating” and “preventing” and the like areto be considered in their broadest context. For example, treatment doesnot necessarily imply that a patient is treated until total recovery.

In the context of this specification, the term “pharmaceuticallyacceptable” means that the compound to which it refers is suitable foruse in contact with tissues of the body without undue toxicity,incompatibility, instability, irritation, allergic response, and thelike, commensurate with a reasonable benefit/risk ratio.

The present invention is predicated on the inventors' surprisingfinding, as exemplified herein, that the use of extracts of variousplants from Pacific islands possess biological activity whenadministered to mammalian skin, promoting bone and cartilage repair andthe formation of new bone and cartilage.

Accordingly, provided herein are biologically active extracts ofHibiscus tiliaceus L. and compositions comprising the same.

While exemplified using extracts of Hibiscus tiliaceus, it is alsocontemplated that the extract may also be derived from one or more ofany Hibiscus sp. including, but not limited to, Hibiscus acetosella,Hibiscus brackenridgei, Hibiscus cannabinus, Hibiscus diversifolius,Hibiscus, lavaterioide, Hibiscus ludwigii, Hibiscus macrophyllus,Hibiscus macropodus, Hibiscus elatus, Hibiscus escobariae, Hibiscusficulneus, Hibiscus paramutabilis, Hibiscus pedunculatus, Hibiscusplatanifolius, Hibiscus radiatus, Hibiscus rosa-sinensis, Hibiscussabdariffa, Hibiscus schizopetalus, Hibiscus scottii, Hibiscussocotranus, Hibiscus sinosyriacus, Hibiscus splendens, Hibiscusstenanthus, Hibiscus striatus, Hibiscus syriacus, Hibiscus trilobus, orHibiscus waimeae.

Extracts and compositions in accordance with some embodiments mayfurther comprise extracts of Vigna sp. and/or Terminalia sp. Inparticular embodiments, the Vigna sp. is Vigna marina (Burm.) and/or theTerminalia sp. is Terminalia catappa.

It is also contemplated that the Vigna sp. extract may be derived fromone or more of, but not limited to, Vigna aconitifolia, Vigna angularis,Vigna caracalla, Vigna debilis, Vigna dinteri, Vigna lanceolata, Vignaluteola, Vigna maritime, Vigna mungo, Vigna o-wahuensis, Vigna parkeri,Vigna radiata, Vigna speciosa, Vigna subterranea, Vigna trilobata, Vignaumbellata, Vigna unguiculata or Vigna vexillate.

It is also contemplated that the Terminalia sp. extract may be derivedfrom one or more of, but not limited to, Terminalia acuminata,Terminalia alata, Terminalia altissima, Terminalia amazonia, Terminaliaangustifolia, Terminalia arborea, Terminalia arbuscula, Terminaliaarchipelagi, Terminalia arjuna, Terminalia australis, Terminaliaavicennioides, Terminalia bellinca, Terminalia bialata, Terminaliabrachystemma, Terminalia brassii, Terminalia bucidoides, Terminaliabuceras {Bucida buceras), Terminalia bursarina, Terminalia calamansanai,Terminalia chebula, Terminalia cherrieri, Terminalia ciliata, Terminaliacitrina, Terminalia copelandii, Terminalia corticosa, Terminaliaeddowesii, Terminalia edulis,. Terminalia elliptica, Terminaliaeriostachya, Terminalia erythrophylla, Terminalia ferdinandiana,Terminalia foetidissima, Terminalia franchetii, Terminalia glabrescens,Terminalia glaucifolia, Terminalia hararensis, Terminalia hecistocarpa,Terminalia intermedia, Terminalia ivorensis, Terminalia januariensis,Terminalia kaernbachii, Terminalia kangeanensis, Terminalia kuhlmannii,Terminalia latifolia, Terminalia mantaly, Terminalia molinetiiTerminalia muelleri, Terminalia myriocarpa, Terminalia nitens,Terminalia novocaledonica, Terminalia oblongata, Terminalia obovata,Terminalia oliveri, Terminalia paniculata, Terminalia parviflora,Terminalia pellucida, Terminalia phanerophlebia, Terminalia phellocarpa,Terminalia prunioides, Terminalia reitzii, Terminalia rerei, Terminaliaschimperiana, Terminalia sericea, Terminalia seriocarpa, Terminaliasubspathulata, Terminalia superba, Terminalia tripteroides or Terminaliavolucris.

Also provided are methods for the promotion or enhancement of bone orcartilage formation, repair or regeneration employing extracts andcompositions as disclosed herein. Such methods find application in,inter alia, the treatment of bone or cartilage injuries, defects ordiseases. The injury, defect or disease may be acute or chronic. Thoseskilled in the art will readily appreciate the scope of injuries,defects and diseases to which the embodiments disclosed herein relate,being those in which the formation of new bone or cartilage, the repairof damaged or otherwise defective bone or cartilage, or the regenerationof bone or cartilage is desirable or advantageous. By way of exampleonly, the injury may be, for example, the result of trauma such as abone fracture or meniscal injury. By way of example, defects anddiseases include congenital bone defects, bone or spinal deformation,osteosarcoma, bone dysplasia, osteoporosis, osteomalacia (rickets),osteogenesis imperfecta (brittle bone disorder), Paget's disease of thebone, osteoarthritis, and other diseases and conditions characterised byor associated with abnormal bone metabolism, formation or resorption.

Embodiments as disclosed herein also find application in the context ofsurgical procedures to correct bone injuries, deformations or diseaseswhere the promotion or enhancement of bone growth or regeneration isadvantageous. For example, the embodiments disclosed herein may be usedin conjunction with a skeletal fusion (e.g. spinal fusion) procedure,spinal disc reconstruction or removal, or the implantation of bonefilling material (such as hydroxyapatite blocks, demineralised bonmatrix plugs, collagen matrices), a fixation device (such as a rod,screw, pin, plate or the like), surgical implant or prosthetic device(such as a prosthetic hip or knee), or with a bone cement (such as thatused to fill a space between a bone and a prosthetic device or to anchora prosthetic device to a bone). The implanted material, implant ordevice may be resorbable or non-resorbable.

In particular embodiments application of extracts and compositions forthe purposes of treating bone injuries, defects and disorders may beachieved using ex vivo procedures. For example, in the case of injuriessuch as bone fractures, a bone fragment(s) or bone segment(s) may beremoved from the subject to be treated and cultured ex vivo with anextract or composition as disclosed herein. The bone fragment(s) or bonesegment(s) and extract or composition may also be cultured together witha bone fixation device or a prosthesis if appropriate. Following cultureunder suitable conditions and for a suitable period of time, to at leastinitiate bone formation, repair or regeneration, the bone fragment(s) orbone segment(s), and optionally the bone fixation device or prosthesismay be reintroduced into the subject. The culture conditions will dependon the application, however suitable conditions can be readilydetermined by a person skilled in the art without the need for undueexperimentation. Alternatively, circumstances may necessitate the use ofbone fragment(s) or segment(s) from other individuals or organisms.

Similarly, osteoblasts or osteoblastic progenitor cells may be culturedex vivo with an extract or composition as disclosed herein prior totheir introduction into a subject in need of treatment. The cells may beautologous (autogeneic), allogeneic or xenogeneic. Ex vivo cell therapymay also be employed using mesenchymal stem cells taken from, forexample, bone marrow or adult peripheral blood, embryonic stem cells,adult stem cells or any other multipotent, pluripotent or totipotentcells, or ‘designer1 cells generated in vitro. The term “osteoblasticprogenitor cells” as used herein encompasses all multipotent,pluripotent and totipotent cells, whether naturally-derived orartificially created, that have the ability to differentiate intoosteoblasts.

Extracts of the invention may be aqueous, oil and/or organic solventbased extracts, obtained by single, combined and/or successiveextraction of any available plant material such as leaves, roots, bark,stems, fruits, shoots, nuts, husks of nuts, seeds, seed capsules,kernels, flowers, vine and/or wood. Suitable extraction processes, andsuitable solvents and liquids for extraction are known to those skilledin the art. Aqueous solvents (for example water, acids, bases); oils(for example oil derived from Cocos nucifera); and organic solvents,which can be polar (such as alcohols for example ethanol), non-polar(for example hexane) and/or halogenated (for example dichloromethane),used for extraction can either be used sequentially for extraction or incombination mixture. Importantly, as exemplified herein, the activity ofthe extract is maintained when extracted into Cocos nucifera oil, polarsolvents (e.g. ethanol) or non-polar solvents (e.g. isopentane).Supercritical fluid extraction using, for example, supercriticalnitrogen or carbon dioxide, may also be used in accordance with theinvention to obtain extracts. Further, it will be appreciated by thoseskilled in the art that an extract of the invention may be subjected toone or more post extraction steps to, for example, increase or maintainthe stability of the extract, modify or change the physical form of theextract or assist in formulating the extract into a composition foradministration to a subject. By way of example only a liquid formextract may be lyophilised to produce a solid form of the extract.

Extracts of the present invention may be derived from any suitable plantmaterial. Suitable plant material includes leaves, roots, bark, stems,fruits, shoots, nuts, husks of nuts, seeds, seed capsules, kernels,flowers, vine or wood. The plant material may be, for example, fresh,dried or freeze dried. For any given plant species more than one plantmaterial may be used for the production of extracts. Where derived fromHibiscus tiliaceus L., typically the extract is a leaf, vine and/or beanextract.

Extract(s) of the invention may be administered in accordance with thepresent invention in the form of pharmaceutical compositions, whichcompositions may comprise one or more pharmaceutically acceptablecarriers, excipients or diluents. Extracts may further be combined withother therapeutic agents for example, but not limited to, antibiotics,antimicrobial agents, antiseptics, anaesthetics, and/or other bone orcartilage growth or repair promoting agents.

It will be understood that the specific dose level of a composition ofthe invention for any particular individual will depend upon a varietyof factors including, for example, the activity of the specificextract(s) employed, the age, body weight, general health and diet ofthe individual to be treated, the time of administration, the stabilityof the extract(s), the site of application on the body, and combinationwith any other treatment or therapy. Single or multiple administrationscan be carried out with dose levels and pattern being determined asrequired depending on the circumstances and the individual to betreated. Suitable dosage regimes can readily be determined by theskilled addressee. A broad range of doses may be applicable. Consideringa human subject, for example, from about 0.1 mg to about 1 mg of extractmay be administered per kilogram of body weight per day. Dosage regimensmay be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered hourly, daily,weekly, monthly or at other suitable time intervals or the dose may beproportionally reduced as indicated by the exigencies of the situation.

Generally, an effective dosage is expected to be in the range of about0.0001 mg to about 1000 mg per kg body weight per 24 hours; typically,about 0.001 mg to about 77 mg per kg body weight per 24 hours; about0.01 mg to about 70 mg per kg body weight per 24 hours; about 0.1 mg toabout 70 mg per kg body weight per 24 hours; about 0.1 mg to about 27 mgper kg body weight per 24 hours; about 1.0 mg to about 27 mg per kg bodyweight per 24 hours. More typically, an effective dose range is expectedto be in the range about 1.0 mg to about 200 mg per kg body weight per24 hours; about 1.0 mg to about 100 mg per kg body weight per 24 hours;about 1.0 mg to about 7 mg per kg body weight per 24 hours; about 1.0 mgto about 25 mg per kg body weight per 24 hours; about 5.0 mg to about 7mg per kg body weight per 24 hours; about 5.0 mg to about 20 mg per kgbody weight per 24 hours; about 5.0 mg to about 15 mg per kg body weightper 24 hours.

The extract may be present in an amount between about 0.001% (w/w) andabout 15% (w/w), or between about 0.001% and about 12%, or between about0.001% and about 10%, or between about 0.005% and about 10%, or betweenabout 0.01% and about 10%, or between about 0.05% and about 10%, orbetween about 0.05% and about 5% in the topical composition.

Compositions as disclosed herein may be administered via any convenientor suitable route such as for example by parenteral, topical, oral, ornasal routes. Parenteral administration may comprise, for example,intraosseous infusion, or intrathecal, intravenous, intraarterial,intramuscular, or subcutaneous administration. In some embodiments,treatment may be effected using methods and compositions as disclosedherein during arthroscopic or open surgical procedures.

Topical formulations typically comprise one or more extracts of theinvention together with one or more acceptable carriers, and optionallyany other therapeutic ingredients. Formulations suitable for topicaladministration may be in any suitable form, formulated for example asliniments, lotions, creams, gels, ointments or pastes. Examples ofpharmaceutically acceptable carriers or diluents are demineralised ordistilled water; saline solution; vegetable based oils such as peanutoil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil,arachis oil or coconut oil; silicone oils, including polysiloxanes, suchas methyl polysiloxane, phenyl polysiloxane and methylphenylpolysolpoxane; volatile silicones; mineral oils such as liquid paraffin,soft paraffin or squalane; cellulose derivatives such as methylcellulose, ethyl cellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols,for example ethanol or isopropanol; lower aralkanols; lower polyalkyleneglycols or lower alkylene glycols, for example polyethylene glycol,polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butyleneglycol or glycerin; fatty acid esters such as isopropyl palmitate,isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar;carrageenan; gum tragacanth or gum acacia, and petroleum jelly.Typically, the carrier or carriers will form from 10% to 99.9% by weightof the compositions.

Lotions according to the present invention include those suitable forapplication to the skin or to an epidermal appendage. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturiser such as glycerol, or oil such as coconut oil,castor oil or arachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the extract for external application. Theymay be made by mixing the extract in finely-divided or powdered form,alone or in solution or suspension in an aqueous or non-aqueous fluid,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as coconut, almond,corn, arachis, castor or olive oil; wool fat or its derivatives, or afatty acid such as stearic or oleic acid together with an alcohol suchas propylene glycol or macrogols.

The compositions of the invention may be included in topical vehicles inan amount between about 0.001% (w/w) and about 90% (w/w), or betweenabout 1% (w/w) and about 7% (w/w), or between about 1% (w/w) and about40% (w/w), or between about 1% (w/w) and about 20% (w/w) or about 0.01%,0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% 14%, 15%,16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 7%, 55%, 17%, 65%,70%, 75%, 80%, 85%, 90%.

The extracts and/or compositions also may be impregnated in any forminto transdermal patches, plasters, and dressings such as bandages orhydrocolloid dressings, for example in liquid or semi-liquid form.

The extracts and/or compositions may also be coated on to or used as anadjunct to a bone fixation device, a prosthesis or a bone cement. Theextracts and/or compositions may also be used as an additive to a bonegraft extender or bone substitute, for example, allograft bone,hydroxyapatite, wollanosite or tricalcium phosphate,

In particular circumstances, for example in the post surgical promotionof bone and cartilage formation, repair or regeneration, administrationof compositions by parenteral administration, typically injection, maybe appropriate. Pharmaceutical forms suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol and liquid polyethylene glycol,and the like), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of superfactants. The preventions of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilisation. Generally, dispersions are prepared byincorporating the various sterilised extract(s) into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and the freeze-drying techniquewhich yield a powder of the extract plus any additional desired extractfrom previously sterile-filtered solution thereof.

The compositions of the invention may also be conveniently presented inunit dosage form and prepared by any of the methods well known in theart of pharmacy. The method may include the step of bringing thecomponents of the oral composition into association with a carrier whichconstitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing intoassociation the components of the oral composition with a liquid carrieror finely divided solid carrier, or both and then, if necessary, shapingthe product into the desired composition.

Compositions suitable for oral administration may be presented asdiscrete units (i.e. dosage forms) such as gelatine or HPMC capsules,cachets or tablets, each containing a predetermined amount eachcomponent of the composition as a powder, granules, as a solution or asuspension in an aqueous liquid or a non-aqueous liquid, or as anoil-in-water liquid emulsion or a water-in-oil liquid emulsion.

When the composition is formulated as capsules, the components of theoral composition may be formulated with one or more pharmaceuticallyacceptable carriers such as starch, lactose, microcrystalline celluloseand/or silicon dioxide. Additional ingredients may include lubricantssuch as magnesium stearate and/or calcium stearate.

Tablets may be prepared by compression or moulding, optionally with oneor more accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the components of the oral compositionin a free-flowing form such as a powder or granules, optionally mixedwith a binder, lubricant (for example magnesium stearate or calciumstearate), inert diluent or a surface active/dispersing agent. Mouldedtablets may be made by moulding a mixture of the powdered compositionmoistened with an inert liquid diluent, in a suitable machine. Thetablets may optionally be coated, for example, with an enteric coatingand may be formulated so as to provide slow or controlled release of thecomposition therein.

The present invention contemplates combination therapies, whereinextracts or compositions as described herein are coadministered withother suitable agents or treatments which may facilitate the desiredtherapeutic effect. For example, one may seek to administer antibiotics,antimicrobial agents, anaesthetics, analgesics, or other bone orcartilage growth- or repair-promoting agents in combination withextracts or compositions disclosed herein. By “coadministered” is meantsimultaneous administration in the same formulation or in two differentformulations via the same or different routes or sequentialadministration by the same or different routes. By “sequential”administration is meant a time difference of from seconds, minutes,hours or days between the administration of the two types of agents. Theagents may be administered in any order. For example, embodiments of thepresent invention contemplate the administration of a lipid or an oil,for example Cocos nucifera oil prior to, or after, the extract orcomposition is administered.

The reference in this specification to any prior publication (orinformation derived from it), or to any matter which is known, is not,and should not be taken as an acknowledgment or admission or any form ofsuggestion that that prior publication (or information derived from it)or known matter forms part of the common general knowledge in the fieldof endeavour to which this specification relates.

The present invention will now be described with reference to thefollowing specific examples, which should not be construed as in any waylimiting the scope of the invention.

EXAMPLES Example 1—Preparation of a Combination Extract of Vigna Marina(Burm.) Merr., Cocos Nucifera L., Terminalia Catappa L. and HibiscusTiliaceus L. in Coconut Oil

Coconut oil (200 mL) was added to fresh shredded leaves of Vigna marina(Burm.) Merr. (−100 g) and fresh shredded leaves of Terminalia catappaL. (100 g). The mixture was placed in a crucible in a water bath at 100°C. for 20 min. The mixture was removed from the heat and immediatelyfiltered and pressed to extract the coconut oil. The resultant coconutoil, containing extracts of Vigna marina (Burm.) Merr. and Terminaliacatappa L, was added to the fresh crushed husk of a green nut of Cocosnucifera L (100 g) and the shaved bark of Hibiscus tiliaceus L. (100 g).The mixture was left to settle for 4 h and then filtered and pressed toextract the coconut oil. The coconut oil was then inverted and stored atless than 20° C. until the coconut oil solidified. Any remainingmoisture or solid in the mixture were removed by decanting from thesolidified coconut oil.

The coconut oil was then heated in a hot water bath at approximately 13°C. and filtered. The resultant filtrate contained a combination extractof Vigna marina (Burm.) Merr., Cocos nucifera L., Terminalia catappa L.and Hibiscus tiliaceus L.

Example 2—Preparation of Extracts of Hibiscus Tiliaceus L 2.1 HibiscusTiliaceus L. Extract in Coconut Oil

The fresh crushed bark of Hibiscus tiliaceus L. (100 g) were immersed incoconut oil (100 mL). The extract was prepared by cold pressing theHibiscus tiliaceus L. and coconut oil mixture; or by heating themixture. The cold pressing was carried out by placing the Hibiscustiliaceus L. and coconut oil mixture inside a new coffee plunger andleaving the mixture to settle for 1 h, the plunger was then depressedthereby extracting the oil. The mixture was left to settle for a further5 min and the plunger depressed again to release more oil. This processwas repeated 5 times until no further oil was obtained. When the extractwas prepared by heating, the Hibiscus tiliaceus L. and coconut oilmixture was heated in a crucible in a water bath at 100° C. for 20 min,removed from the heat, and then immediately filtered and pressed toextract the coconut oil. Any remaining moisture or solid in the extractafter cold pressing or heating and filtering were removed by invertingthe coconut oil extract and storing it at less than 20° C. until thecoconut oil solidified and then decanting the solidified coconut oil; orby decanting in a separating funnel.

2.2 Hibiscus Tiliaceus L. Extract in Coconut Oil Following Extraction inEthanol

The fresh crushed bark of Hibiscus tiliaceus L. (1000 g) were immersedin 95-100% ethanol (2000 mL) and left to steep for 6 h. The ethanol wasthen removed and the Hibiscus tiliaceus L. steeped in a second aliquotof ethanol (2000 mL) for a further 6 h. The resultant ethanol extractswere added to coconut oil (27 mL). More than half the volume of ethanolwas removed by heating above the boiling point of ethanol. The coconutoil was then extracted by decanting from the solution following standingfor at least 2 h in a decanting vessel; or by inverting the coconut oilextract and storing it at less than 20° C. until the coconut oilsolidified and then decanting the solidified coconut oil.

2.3 Hibiscus Tiliaceus L. Extract in Coconut Oil Following Extraction inEthanol and Hydrocarbon

The fresh crushed bark of Hibiscus tiliaceus L. (1000 g) were immersedin 95-100% ethanol (2000 mL) and left to steep for 6 h. The ethanol wasthen removed and the Hibiscus tiliaceus L. steeped in a second aliquotof ethanol (2000 mL) for a further 6 h.

The resultant ethanol solution was agitated with hexane, pentane, methylbutane or an equivalent hydrocarbon or fluoro/chloro/bromo-hydrocarbon(27 mL). The hydrocarbon layer and oily foam layer were decanted fromthe solution following standing for at least 2 h in a decanting vessel;or by the dilution of the ethanol solution from 95% to a lowerconcentration, nominally but not necessarily 7% ethanol and thendecanted after settling of the layers. Water (27 mL) or alcohol (27 mL)was added to the extracted hydrocarbon layer. To this coconut oil (27mL) was added and the mixture agitated and allowed to settle. Thehydrocarbon was removed from heating the mixture to the boiling point ofthe hydrocarbon.

2.4 Hibiscus Tiliaceus L. Extract in Coconut Oil Following Extraction inEthanol

The fresh crushed bark’ of Hibiscus tiliaceus L. (1000 g) were immersedin 95-100% ethanol (2000 mL) and left to steep for 6 h. The ethanol wasthen removed and the Vigna marina (Burm.) Merr. steeped in a secondaliquot of ethanol (2000 mL) for a further 6 h. The resultant ethanolsolution was agitated with coconut oil (27 mL).' The hydrocarbon wasdecanted from the solution following standing for at least 2 h in adecanting vessel; or by the dilution of the ethanol solution from 95% toa lower concentration, nominally but not necessarily 7% ethanol and thendecanted after settling of the layers. The decanting of the coconut oilmay be facilitated by the addition of a low boiling point hydrocarbon tothe mixture as it agitates and settles. The coconut oil was theninverted and stored at less than 20° C. until the coconut oilsolidified, or by decanting in s separation vessels.

Example 3—Effect of Individual Extracts of Hibiscus Tiliaceus L. on Skin

An assessment of the activity of the extracts prepared as described inExample 2 on skin was carried out. A daily dose of 1 mL of Hibiscustiliaceus L. extract in coconut oil (Example 2.1) and 1 mL of Hibiscustiliaceus L. extract in ethanol (Example 2.2). The extracts were appliedtopically to the epithelial surface of the backs of 10-14 week oldfemale rats. The controls were 10-14 week old female rats treated dailywith 1 mL of coconut oil or left untreated.

FIGS. 1A to 1D show the histology of the skin at 7 days of the untreatedcontrol (1A) after a daily application of coconut oil (1B); an extractpreparation of Hibiscus tiliaceus L. in ethanol (1C); and coconut oilfollowing extraction with ethanol (1D). The histological profiles showthat the extract has limited activity in ethanol, but that in coconutoil the extract induces hypertrophy of the epithelium and the epithelialappendages in particular hair follicles.

An assessment of the activity of the extracts prepared as described inExamples 1 and 2.2 on skin was carried out. A daily dose of 1 mL of theextracts were applied topically to the epithelial surface of the backsof 12 week old. female New Zealand rabbits. The control was theuntreated skin of 12 week old female New Zealand rabbits. FIGS. 2A to 2Fshow the histology of the skin at 7 days of the untreated control (2Aand B); and after a daily application of a combination extract of Vignamarina (Burm.) Merr., Cocos nucifera L., Terminalia catappa L. andHibiscus tiliaceus L. in coconut oil prepared as described in Example 1(2C and D); and Hibiscus tiliaceus L. in coconut oil prepared asdescribed in as Example 2.2 (2E and F). The histological profiles showthat the extracts induce hypertrophy of the epithelium and theepithelial appendages in particular hair follicles.

Example 4—Treatment of Fractures in Oestrogen and Non-OestrogenDeficient Bones

A study was carried out to assess the effect of an extract of Hibiscustiliaceus L. prepared as described in Example 2.1 on bone fracturerepair in oestrogen deficient and non-oestrogen deficient rats. In thestudy a daily dose of 1 mL of the extract was applied topically to theepithelial surface of 18 month old, oestrogen deficient (ovaries removedat age 6 weeks) rats and 18 month old rats with their ovaries intact (acontrol surgical procedure was carried out at age 6 weeks and the ratsallowed to recover), following a surgically created fracture of thefemur by osteotomy, and operative repair with internal fixation. Theextract was applied daily to the test animals for a total of 3 weeks.Control animals were 18 month old, oestrogen deficient (ovaries removedat age 6 weeks) rats and 18 month old rats with their ovaries intactwith no topical application of the extract. At 3 weeks the histology ofthe animals was assessed, with particular attention paid to thecartilage and new bone formation in the healing fracture.

FIGS. 3A to 3H show the histology at the fracture site. The resultsindicate extensive new cartilage growth and new bone formation followingthe cartilage caps in the treated groups, particularly of note is theamount occurring in the oestrogen deficient bones.

FIG. 4 is a graph showing histological scoring of the fracture sites ofuntreated -oestrogen deficient rats (Ovx Control) and treated oestrogendeficient rats (Ovx rx) rats. In order to determine the totalhistological scoring the following fracture healing and tissue responseparameters were scored: callous formation, bone union, marrow changesand cortex remodelling. Each parameter was scored from 0 (no callousformation; no new bone in the fracture; fibrous tissue or red; and noremodelling) to 3 (full callous formation across the defect; full bonebridge union; adult type fatty marrow; and full remodelling cortex),with a score of 1 indicating mild healing (<7%) and a score of 2indicating moderate healing (>7% but less than full fracture healing ortissue response). After 3 weeks there was a significantly higher totalhistological score for the fracture healing and tissue response at thefracture sites of treated oestrogen deficient rats compared to untreatedoestrogen deficient rats.

Example 5—Treatment of Fractures

A study was carried out to assess the effect of an extract of Hibiscustiliaceus L. prepared as described in Examples 1 and 2.1 on bonefracture healing. In the study a daily dose of 1 mL of the extract wasapplied topically to the epithelial surface of 12 week old NZ whiterabbits, following a surgically created 20 mm defect of the ulna(including periosteum) by osteotomy, and with no graft or substitutefiller. The extract was applied daily to the test animals for a total of1 week. Control animals were 12 week old NZ white rabbits with notopical application of the extract. At 1 week radiology and histology ofthe animals was assessed, with particular attention paid to cartilageand new bone formation.

FIGS. 5A to 5D are radiographs of the bone and FIGS. 6A to 6F and 7A to7D show histology of the bone defect at the fracture site. The figuresillustrate the effect of the extracts of Hibiscus tiliaceus L. on thehealing of the fracture. After seven days there was significantly morebone formation, new cartilage formation and greater vascularity at thefracture site than in untreated or control treatment fractions.

FIG. 8 is a graph of the bone growth score which shows the bone healingof the defect evaluated using a healing score definition based on thepercent of bone or cartilage occupied space. The following scoringsystem was used: 0=no visible bone; 1=minimal (1% to 20%) new bohegrowth no cartilage; 2=mild (1 to 20%) cartilage with new bone growth;3=moderate (20% to 40%) cartilage and bone growth; 4=marked (17 to 80%)cartilage and bone growth; 5=complete defect filled with new bone andcartilage; 6=defect filled with new bone and no cartilage; 7=corticalbone replacing woven bone; and 8=new marrow space forming. After 7 daysthere was significantly more bone formation at the fracture site treatedwith Hibiscus tiliaceus L. in coconut oil following extraction withethanol (eAo) than the fracture sites treated with Hibiscus tiliaceus L.precipitate of intermediate density in ethanol (HeA Intermediate) inuntreated or control treatment fractions; Hibiscus tiliaceus L. inethanol (eA Ethanol); or a combination extract of Vigna marina (Burm.)Merr., Cocos nucifera L., and Terminalia catappa L. in coconut oilfollowing extraction with ethanol (eNPKo).

Example 6—Bone Formation Following a Spinal Fusion Autograft

A study was carried out to assess the effect of an extract of Hibiscustiliaceus L. prepared as described in Example 2.4 on bone fracturerepair. In the study a daily dose 1 mL of the extract was appliedtopically to the epithelial surface of 12 week old NZ white rabbits,following anaesthetisation and a bilateral single level postero-lateralfusion using autograft harvested from the iliac crests. The extract (5mL) was applied daily to the test animals for 5 days and then twiceweekly until sacrifice. The control animals were 12 week old NZ whiterabbits with no topical application of the extract.

FIGS. 9A to 9E show x-rays and a CT scan of the spine in treated anduntreated controls. The Figures show the effect of the extracts ofHibiscus tiliaceus L. on bone growth surrounding the site of theautograft. After only seven days there was more bone formation, newcartilage formation and greater vascularity at the fracture site than inuntreated or control treatment fractures.

FIGS. 10A and 10B show the histology of the spine at 6 weeks. FIG. 10Ashows the histology of the control autograft at 6 weeks, stained withHaematoxylin and Eosin covering the entire spinal fusion mass. Thetransverse processes can be seen at each end of the mass with the wellformed marrow spaces and dense cortical bone (A) and adjacent to thetransverse process is a small amount of cartilage (B). The bonefragments from the autograft are still visible and are necrotic, withthe loss of lacunae (C). The centre of the fusion mass is predominantlyfibrous tissue (D) and some new woven bone (E).

FIG. 10B shows the histology of the treated autograft at 6 weeks,stained with Haematoxylin and Eosin covering the entire spinal fusionmass. The transverse processes can be seen at each end of the mass withthe well formed marrow spaces and dense cortical bone (A) and adjacentto the transverse process is corticated new bone (B). The bone fragmentsfrom the autograft are barely visible and are mostly absorbed andreplaced (C). The centre of the fusion mass is predominantly cartilage(D), new marrow spaces (E) and some new woven bone (F).

FIG. 11 show the results of mechanical testing of the treated autograftat 8 weeks compared to untreated controls at 12 weeks. The ligaments andinter-vertebral discs were surgically cut in all spines leaving thetransverse process spinal fusion mass as the bridge between thevertebral bodies. Loading was applied until the fusion mass broke. Thepeak load was recorded for each sample. The data shown in FIG. 11suggests that the 8 week treated group is at least as strong as the 12week control with a trend towards greater bone strength.

Example 7—Analysis of Extracts of Hibiscus Tiliaceus L

A method of investigating the composition of the extracts was developedusing gas chromatographic-mass spectrometric (GC-MS) analysis. Forexample, GC-MS analysis was carried out on an extract of Hibiscustiliaceus L. in coconut oil following extraction in ethanol andhydrocarbon (Example 2.3) and the hydrocarbon intermediate layer.

FIG. 12A shows mass chromatograms of an extract of Hibiscus tiliaceus L.in coconut oil with a main peak at 30.99 min. The intermediate layeralso contained a peak at 30.99 min (FIG. 12B), however, the peak was atleast seven times less than that observed in the extract of Hibiscustiliaceus L. in coconut oil. In summary, the dominant peaks in the masschromatograms of the extract of Hibiscus tiliaceus L. in coconut oilwere at 30.99, 35.47, 37.48, 39.13, 1.85, 43.28, 49.04, 52.51, 55.97 and58.54 min. The majority of the smaller peaks correspond to ethyl estersof fatty acids found in coconut oil.

The invention is claimed as follows:
 1. A biologically active extract ofHibiscus tiliaceus L., wherein the extract is prepared using and/orcomprises a plant based oil.
 2. The extract of claim 1, wherein theextract is derived from one or more of a bark, leaf, vine, bean, husk ornut.
 3. The extract of claim 1, wherein the Hibiscus tiliaceus L.extract is derived from bark.
 4. The extract of claim 1, wherein theplant based oil is derived from Cocos nucifera.
 5. The extract of claim4, wherein the Cocos nucifera oil is virgin Cocos nucifera oil, refinedCocos nucifera oil, hydrogenated Cocos nucifera oil or fractionatedCocos nucifera oil.
 6. A method for promoting the formation, repair orregeneration of bone or cartilage in a subject in need thereof, themethod comprising administering to the subject an effective amount of(a) a composition comprising a biologically active extract of Hibiscustiliaceus L., wherein the extract is prepared using and/or comprises aplant based oil, and one or more pharmaceutically acceptable carriers,diluents and/or excipients, or (b) osteoblasts, osteoblastic progenitorcells, or bone segments or fragments cultured in the presence of thecomposition (a).
 7. The method of claim 6, further comprisingadministering to the subject an effective amount of an extract of one ormore of Vigna marina (Burm.) Merr., Cocos nucifera L., or Terminaliacatappa L.
 8. The method of claim 6, wherein the subject is sufferingfrom a bone or cartilage injury, defect or disease, or may besusceptible or predisposed to a bone or cartilage injury, defect ordisease, or is undergoing or has undergone a bone or cartilage graftprocedure.
 9. A method for treating a bone or cartilage injury, defector disease in a subject, the method comprising administering to thesubject an effective amount of (a) a biologically active extract ofHibiscus tiliaceus L., wherein the extract is prepared using and/orcomprises a plant based oil, (b) a composition comprising a biologicallyactive extract of Hibiscus tiliaceus L., wherein the extract is preparedusing and/or comprises a plant based oil, and one or morepharmaceutically acceptable carriers, diluents and/or excipients, or (c)osteoblasts, osteoblastic progenitor cells, or bone segments orfragments cultured in the presence of the extract (a) or the composition(b).
 10. The method of claim 9, wherein the extract or compositionenhances the rate or extent of healing of the injury, defect or disease.11. The method of claim 9, wherein the extract or composition promotesbone or cartilage formation, repair or regeneration.
 12. The method ofclaim 9 for promoting bone or cartilage growth surrounding a bone orcartilage graft or implantable device, wherein the composition orosteoblasts, osteoblastic progenitor cells or bone segments or fragmentsare administered to the subject prior to, during or after a graft orimplantation procedure.
 13. The method of claim 12, wherein the graft isan autograft or allograft.
 14. The method of claim 12, wherein the graftor implantation procedure is for the repair of a bone fracture or forthe purposes of skeletal fusion.
 15. The method of claim 12, wherein theextract, the composition, or the osteoblasts, osteoblastic progenitorcells or bone segments are coated onto the implantable device, used asan adjunct to the device, or used as an adjunct to a bone cement used toanchor the device.
 16. The method of claim 9, further comprisingadministering to the subject an effective amount of an extract of one ormore of Vigna marina (Burm.) Merr., Cocos nucifera L., or Terminaliacatappa L.
 17. The method of claim 16, wherein the extract of Vignamarina (Burm.) Merr., Cocos nucifera L., or Terminalia catappa L. isformulated in the same composition as the extract of Hibiscus tiliaceusL.
 18. The method of claim 9, wherein the administration to the subjectis during an arthroscopic or open surgical procedure.
 19. The method ofclaim 9, wherein Cocos nucifera oil is administered prior to or afterthe extract or composition is administered.